Delta-like gene expressed in neuroendocrine tumors

ABSTRACT

A polynucleotide molecule dlk is expressed in neuroendocrine tumors, including small cell lung carcinoma. A Dlk polypeptide encoded by dlk polynucleotide molecule can be used in detecting the existence of a primary or secondary neuroendocrine tumor. Monoclonal antibodies are produced against Dlk which are useful for detection and therapy of a neuroendocrine tumor.

This application is a divisional of application Ser. No. 07/989,537,filed Dec. 11, 1992, abandoned.

BACKGROUND OF THE INVENTION

The expression of genes during the development of a pluripotent orprogenitor cell into a differentiated, mature cell can provide a contextfor the study of tumorigenic cells whose origin is derived from suchprogenitor cells. In certain hematopoietic or epithelial tumors,malignant gene expression correlates substantially with the expressionobserved during normal development of the tissue from which the tumororiginates, Gordon et al., J. Cell Biol. 108: 1187 (1989); Godal et al.,Adv. Cancer Res. 36: 211 (1982). In fact, many biological activities ofprogenitor cells, including cellular migration and tissue remodeling,resemble pathological activities of cancer cells, such as metastases andtumor invasion.

Neuroblastoma, a tumor of the adrenal gland which afflicts personsduring early childhood, is another system in which tumor biologycorrelates with that of normal differentiation and morphogenesis of itsprogenitor cells (neuroblast). Neuroblastoma is an embryonal tumor thatexhibits both undifferentiated and differentiated histopathology. Thedevelopment of neuroblastoma tumors mimics stages identifiable duringhistogenesis of its tissue of origin, the adrenal medulla. Cooper etal., Cell Growth and Diff. 1: 149 (1989).

During the development of human adrenal medulla neuroblast into maturechromaffin cells, four individual genes are expressed in a sequentialpattern. Once a neuroblast is induced to differentiate along aneuroendocrine pathway, the progressive stages of chromaffin maturationare marked by a temporal expression of genes denoted TH, CGA, pG2 andB2M (Cooper, supra. at page 153). Cooper identified that the pattern ofgene expression of these four markers in neuroblastoma cells mimics thatof normal adrenal neuroblast arrested during three different stages ofdevelopment.

One of these marker genes, pG2, was identified first inpheochromocytoma, a tumor of the adult adrenal medulla (Helman et al.,PNAS USA 84: 2336 (1987)). Helman reported that pG2 also is highlyexpressed normal human adrenal cells.

Helman isolated a full-length cDNA from a human adrenal cDNA library,and identified a corresponding pG2 protein containing 286 amino acids,having a predicted molecular weight of 30,600 daltons (Helman et al.,Nucleic Acids Res. 18(3): 685 (1990)).

A gene having developmentally-regulated expression, paralleling that ofpG2, would be useful for detecting pheochromocytoma or neuroblastoma bygenetic methods, especially since pG2 expression is restricted to theadrenal gland in non-malignant tissues.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide anewly-isolated polynucleotide molecule, dlk, which can be employed ingenetic assays to provide a method for detection of a primary orsecondary pheochromocytoma or neuroblastoma, or identification of astage of these tumors.

It is also an object of the present invention to provide a method fordetecting primary or secondary small cell lung carcinoma (hereafter,SCLC) or for staging tumor progression of SCLC, which employs dlkpolynucleotide molecule in genetic assays.

It is a further object to provide a polynucleotide molecule, designateddlk, which encodes a corresponding Dlk polypeptide. Dlk polypeptide isuseful for generating monoclonal or polyclonal antibodies havingspecificity for an epitope of Dlk polypeptide.

Dlk-specific antibodies, and in particular, labeled monoclonalDlk-specific antibodies, are useful for detection of primary orsecondary neuroendocrine tumors. According to the present invention,Dlk-specific monoclonal antibodies conjugated to a toxin are useful fortreatment of primary or secondary neuroendocrine tumors, as well.

In accomplishing these and other objects of the invention, there hasbeen provided, in accordance with one aspect of the present invention,an isolated polynucleotide molecule comprising a DNA sequence encoding aDlk polypeptide.

An object of the present invention is to provide an isolated Dlkpolypeptide consisting essentially of the amino acid sequence shown inFIG. 1B (SEQ ID NO:2), or in FIG. 1A (SEQ ID NO:1).

Another object of the present invention is to provide an isolatedpolynucleotide molecule which encodes a human or mouse Dlk polypeptideconsisting essentially of the amino acid sequence shown in FIGS. 1B (SEQID NO:2), or FIG. 1A (SEQ ID NO:1), respectively.

A further object of the invention is to provide a method for detecting atumor which expresses dlk, including the steps of contacting a samplesuspected of being tumorigenic with dlk polynucleotide molecule, underconditions permissive of hybridization between dlk polynucleotidemolecule and the sample, and detecting hybridization between thepolynucleotide molecule and sample.

Yet another object of the invention is to provide a method for detectinga small cell lung carcinoma, including the steps of contacting a sampleof bronchial epithelial cells suspected of being tumorigenic with dlkpolynucleotide molecule, under conditions permissive of hybridizationbetween dlk polynucleotide molecule and the sample, and detectinghybridization between the polynucleotide molecule and sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of (A) mouse and (B) human Dlk amino acidsequences (SEQ ID NOS:1 and 2 respectively). Identical amino acids areshown by the character (.linevert split.). Similar amino acids areindicated by ( ) and classified into the following groups: A, S & T; D &E; N & Q; R & K; I, L, M & V; and F, Y & W. Potential biologicallysignificant sites, found in the database PROSITE (accessiblecommercially through Intelligenetics Inc. (Mountain View, Calif.)), areindicated by numbers: 1. N-glycosylation site; 2. Protein Kinase Cphosphorylation site; 3. N-myristylation site; 4. Aspartic acid andasparagine hydroxylation site. Potential sites of cleavage in the signalpeptide are indicated by (*).

FIG. 2 shows human dlk DNA sequence (SEQ ID NO:3).

FIG. 3 shows mouse dlk DNA sequence (SEQ ID NO:4).

FIG. 4 shows an alignment of a consensus sequence of dlk EGF-likerepeats with EGF-repeats from several invertebrate homeotic genes. Asdescribed in Example 3, a dlk EGF-like repeat consensus sequence (SEQ IDNOS:6 and 7) was obtained by alignment of 12 EGF-like repeats of dlkfrom both human and mouse. This consensus sequence then was aligned withthe consensus sequences of several invertebrate homeotic genes(similarly obtained) and mouse EGF. SEQ ID NO:5 is also shown in thisfigure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A human polynucleotide molecule, dlk, and a corresponding humanpolypeptide, Dlk, encoded by dlk, were discovered, isolated andcharacterized. Human dlk polynucleotide molecule is expressed inpheochromocytoma, neuroblastoma, and SCLC tumors.

Dlk protein is about 383 amino acids in length and has a molecularweight of about 42,000 daltons. In addition to human dlk, otherpolynucleotide molecules belonging to the dlk family are providedaccording to the invention, including murine dlk (FIG. 3, SEQ ID NO:4)and a human variant-dlk, isolated from placenta as described herein.

According to the present invention, isolated polynucleotide molecules orfragments thereof belonging to the dlk family are useful to detect SCLCand neuroendocrine cancers. The expression patterns of dlk can beexploited both (1) to detect primary or secondary tumor cells by thepresence of dlk and (2) to diagnose the stage of a tumor that expressesdlk, by measuring the level of dlk expression.

Dlk is a transmembrane protein having expression pattern, in normalnon-fetal tissues, which is restricted to the adrenal gland. As aconsequence, Dlk is a readily accessible target for antibody imaging ortherapy of SCLC, pheochromocytoma and neuroblastoma tumors. According tothe present invention, antibodies having specificity for Dlk protein aremade and employed to detect or treat cells which produce Dlk protein.

Human dlk cDNA comprises a polynucleotide molecule having the sequenceshown in FIG. 2 (SEQ ID NO:3), as determined by nucleotide sequenceanalysis. The open reading frame, nucleotides 174(ATG) - 1322(TAA), is1149 nucleotides long. Mouse dlk polynucleotide molecule comprises a DNAsequence (SEQ ID NO:4) having an open reading frame, nucleotides134(ATG) - 1288(TAA), of 1155 nucleotides, as shown in FIG. 3. MurineDlk protein is about 385 amino acids and has a molecular weight of about42,000 daltons.

According to the present invention, a variant of human Dlk is identifiedin which an amino acid is deleted. A cDNA encoding "variant-Dlk," inwhich amino acid number 347 of the amino acid sequence shown in FIG.1(B) (SEQ ID NO:2) is deleted, was isolated from a total human placentalcDNA library. Missing amino acid number 347 is located in anintracellular domain of the protein. The placental library containingvariant-Dlk also contained substantial amounts of the non-variant form,that is, dlk polynucleotide molecule shown in FIG. 1B (SEQ ID NO:2).

dlk polynucleotide molecule was identified by examination of cDNAexpression products of human SCLC (hSCLC) lines which were responsive tostimulation with the ligand, gastrin-releasing peptide (GRP), aneuropeptide implicated in the release of gastrin through itsinteraction with a G-protein-coupled receptor, GRP receptor. GRP(peptide) is a mitogen for normal lung epithelial and SCLC cells, andfor murine Swiss 3T3 fibroblasts.

GRP-responsive hSCLC lines were compared with murine fibroblast celllines that were differentially responsive to GRP. This approach, asdetailed in Example 1, yielded a partial length cDNA molecule whichhybridized with a 1.6 KbmRNA expressed both in responsive fibroblastsand responsive SCLC lines. A commercial library of Swiss 3T3 fibroblastswas screened with the partial length cDNA, which yielded several cloneshaving 1.6 Kb inserts, which then were sequenced.

A computer search of the databases "Swissprot" and "NBRF Protein,"described by Devereux et al., Nuc. Acids Res. 12(1): 387 (1984),indicated a high degree of homology between Dlk and proteins encoded byseveral homeotic genes, identified in Example 3. Homeotic genes aredevelopment-controlling regulatory genes that assign spatial identity togroups of cells with respect to their morphogenic fates. In segmentedorganisms, for example, homeotic genes are required for the propermorphogenesis of a distinct region (such as a leg, or antennae) and actby controlling the activities of other genes during development. Dlkprotein of the present invention exhibited highest homology with theprotein Delta, a neurogenic locus involved in normal neuraldifferentiation in Drosophila. Thus, the present protein was designated"Dlk" for being "delta-like."

Mouse and human Dlk protein sequences share 86.2% identity as well asmany potential sites of biological importance, including 6 epidermalgrowth factor (EGF)-like repeats, a transmembrane domain, and a signalpeptide domain at the amino terminus. Based upon these structuralfeatures, dlk appears to be a new member of the family of EGF-likeneurogenic genes of Drosophila, which are involved in developmentaldecisions of the embryonal ectoderm to differentiate into epidermal orneuronal cells.

The expression pattern of dlk and its sequence homology with homeoticproteins support that dlk functions in the differentiation decisionstaken by the cells of the chromaffin lineage. As detailed in Example 2,dlk is expressed in primary and secondary pheochromocytoma andneuroblastoma, and in normal (non-histopathological) human adrenalmedulla and placental cells. According to the present invention, SCLCand neuroblastoma are the only tumors known to express dlk as a functionof differentiation.

An isolated dlk, dlk-variant, and murine dlk polynucleotide and proteinproducts are employed in diagnostic methods (described further below)and are made according to the following description. Hereafter, thetechniques and applications described for dlk polynucleotide molecule(DNA, RNA) and Dlk protein are intended to be useful for DNA, RNA andprotein of murine dlk, and of variant-dlk, as well.

A Dlk polypeptide, according to the present invention, is produced byrecombinant DNA techniques, such as those set forth generally byManiatis et al., MOLECULAR CLONING--A LABORATORY MANUAL, Cold SpringHarbor Laboratory (1982). Methods specifically suitable to cloning andthe dlk polynucleotide molecule are described in Example 1.

The dlk polynucleotide molecule of FIG. 1 (SEQ ID NO:1) can be clonedinto suitable expression vectors and expressed in prokaryotic, insect oreukaryotic expression systems, including Baculovirus or E. coli. Forinstance, the protein EGF, having similar functional domains as Dlk, wasexpressed in E. Coli (Boehringer Manheim). With conventional techniques,therefore, a sequence encoding a Dlk protein, can obtained as a cDNAfrom mRNA from a commercial adrenal medulla or Swiss 3T3 fibroblastlibrary, or from SCLC, neuroblastoma or pheochromocytoma cell lines. ThemRNA can be converted to double-stranded DNA using cDNA cloningtechniques well-known to the art, including PCR-based techniques.Linkers or tails may be placed on the ends of the double-stranded DNA toprovide convenient restriction sites. After restriction digestion, theDNA may be introduced to any site in a vector, such as a plasmid vector,which has been restricted with a restriction enzyme that generatescompatible ends. A suitable plasmid vector in this context is pGEX-λ(Pharmacia). Following ligation, by means of standard techniques, theDNA is introduced into a cell, where its expression produces the desiredprotein.

Alternatively, a Dlk polypeptide is produced using a commerciallyavailable in vitro translation kit from NEN (Boston, Mass.), as detailedin Example 1. This kit employs a translation system (includingribosomes, polymerases, amino acids, etc.) derived from rabbitreticulocyte lysates to express dlk mRNA.

The term "isolated" is used in connection with dlk polynucleotidemolecule to indicate that such a molecule is free of proteins with whichit is normally associated, such as histones. An isolated form of the dlkis substantially free of other DNA that does not function to regulate,promote, enhance or otherwise modulate its expression.

The term "isolated," with reference to Dlk protein, connotes apolypeptide that is free of other proteins with which it is normallyassociated.

An isolated dlk polynucleotide molecule is useful in detection ofprimary SCLC and in identifying metastatic spread of SCLC and otherneuroendocrine cancers. More specifically, a method of tumor detectionis provided by the present invention which includes the steps ofcontacting a sample suspected of containing a tumor with a dlkpolynucleotide molecule, and detecting expression of dlk polynucleotideproducts (DNA, RNA, mRNA) in non-adrenal cells. Detection of a dlkpolynucleotide product diagnoses the cells as metastatic cells(secondary tumor) of neuroblastoma, pheochromocytoma or SCLC, or as aprimary tumor of SCLC.

In addition to tumor detection, accomplished by detection ofdlk-expressing cells, the identity of a detected tumor is determined.After recognition of dlk-expression, a tumor type is determined bydetecting a tumor-specific marker, tumor-specific morphology, or bypresentation by the patient of a clinical pathology that is distinctlyassociated with any of the tumors selected from the group includingneuroblastoma, pheochromocytoma or SCLC. For example, information suchas the identification of a cellular marker, histological feature ordisease symptom which is specific to one of the tumors of neuroblastoma,pheochromocytoma or SCLC, is recognized.

If dlk expression is detected in cells of a sample taken from bronchialepithelial tissue or tissue removed from the lung, the detectionidentifies the existence of a primary SCLC. It is preferred that asecond step of confirming the origin of the detected dlk-expressingtumor cells as SCLC be performed by detection of a marker, histologicalfeature, or presentation of a distinctive symptom associated with thistumor. For example the histology of an "oat cell" commonly identifiedwith SCLC is detected to confirm the presence of SCLC.

dlk expression is detected by hybridization with dlk polynucleotidemolecule. This method includes the steps of contacting a samplesuspected of being tumorigenic with dlk polynucleotide molecule anddetecting the presence of hybridization between the polynucleotidemolecule and sample. A positive hybridization indicates that the sampleis tumorigenic.

The polynucleotide molecule or "dlk probe" used to hybridize to dlkexpressed in the sample is a labeled fragment of dlk, or preferably afull-length dlk DNA molecule which will hybridize to mRNA or DNA fromnormal adrenal and neuroendocrine tumor cells. Probes complementary todlk are prepared by conventional methods, and are preferably allowed tohybridize to mRNA or DNA, using conventional in situ techniques, to asample (embedded on a microscope slide by means of a standard fixative).Unhybridized probe is removed by nuclease.

In situ techniques which are known in the art may employ the use offluorescent and radiolabels which can be easily quantitated byfluorescence microscopy or autoradiography, respectively. Generally,fluorescent labels will be preferred. Another labeling technique mayemploy enzymatic tags which generate readily quantifiable colorimetricor chemiluminescent signals. The intensity of hybridization detectedreflects the amount of dlk within the cells of the tissue.

RNA ("Northern") blotting is employed using a dlk polynucleotidemolecule of the invention. According to this method, RNA is isolatedfrom tissue by any of a number of standard procedures (Lehrach, H.,Biochemistry, 16: 4743 (1975)). RNA is subjected to denaturing gelelectrophoresis and transferred to nitrocellulose or other supportmatrix. The dlk mRNA can be detected by hybridization of radioactivelyor non-radioactively labelled dlk, or dlk fragments, preferably underhigh stringency conditions, such as recognized by a scientist in thisfield. The amount of hybridization can be quantified by densitometricmethods.

In yet another embodiment of the present invention, the polymerase chainreaction ("PCR") is used to detect dlk DNA or mRNA in a sample. Toperform PCR, a pair of dlk sequence specific primers is employed, whichhybridize to opposite strands of the dlk gene at offset positions on thedouble helix. Such primers, taken from the dlk polynucleotide sequencesprovided in accordance with the invention, represent fragments whichpreferably are unique to dlk, e.g. sequences having low homology withother proteins than Dlk. Two exemplary dlk-specific primer sequencesuseful in this context include the following sequences (SEQ ID NOS:8 and9 respectively), which encode a portion of the intracellular region ofDlk:

5'-CAA GCC CGA GTT CAC AGG TC-3'

5'-TCG GGG AAG ATG TTG AC-3'.

Other such primer pairs can be selected and utilized, as well.

The primers provide initiation points for DNA synthesis. In the presenceof DNA polymerase, the four nucleotide triphosphates ("NTPs") and othernecessary co-factors, all of which are well known to the art, new DNAstrands are synthesized complementary to the templates which hybridizedwith the primers. Several rounds of synthesis are carried out, withallowance for denaturation of the double stranded products betweenrounds. Preferably, a thermal stable DNA polymerase is used so that itis not necessary to add enzyme anew for each round of synthesis.

The PCR produces a double stranded DNA amplification product which hasthe same sequence as the original stretch of the dlk DNA defined by theends of the primer pair sequences. The amount of PCR product indicatesthe amount of dlk DNA or dlk mRNA in the sample. The product can bedetected by a variety of methods well-known in the art. Where suchproducts are produced in a test tube, or the like, they can be resolvedby agarose or polyacrylamide electrophoresis and detected byfluorescence staining, such as ethidium bromide. Alternatively, one ofthe NTPs may be labelled and the PCR products may be determined bymeasuring incorporation of the labeled NTP. A variety of other methodsfor resolving, detecting and measuring the amount of PCR product arewell-known to the art that are suitable for use in the presentinvention.

PCR may be rendered specific for dlk DNA or dlk mRNA in situ and inliquid PCRs. For instance, RNAse or DNAse may be used to remove onetemplate or the other from the sample, and the use of primers thatdistinguish between the gene and the message, for example, a primer thathybridizes to a sequence in the untranscribed region of the promoterwill be gene specific.

Other techniques suitable to the claimed methods are readily apparent tothe skilled artisan and can include Nuclease Protection Assays, ELISAand Western blotting. Several assay techniques which are based uponimmunological reactions between antigens and antibodies are contemplatedby the invention. In particular, assays which use antibodies havingspecificity for Dlk protein are useful to detect cells which produce Dlkprotein.

Antibodies having specificity for Dlk-expressing cells are obtained bystimulating the immune system of an animal with Dlk protein. In thiscontext, the term "antibody" encompasses monoclonal and polyclonalantibodies. Such an antibody can belong to any antibody class (IgG, IgM,IgA, etc.). According to the present invention, an entire Dlkpolypeptide is injected into an animal for the purpose of obtainingpolyclonal antibodies, or for obtaining lymphocytes or spleen cells forproduction of monoclonal antibodies.

The general techniques of monoclonal antibody (Mab) production, such asdescribed by Kohler and Milstein, Nature 256:495 (1975), are applied toproduce a monoclonal antibody having specificity for Dlk protein. Thisprocedure includes the steps of isolating lymphocytes of an animal whichhas been sensitized or injected with Dlk polypeptide, fusing them withmyeloma cells to produce hybridomas, then screening the hybridomas forproduction of "anti-Dlk antibodies" which bind preferentially to orexhibit binding specificity for Dlk polypeptide.

"Antibody" also encompasses fragments, like Fab and F(ab')₂, of anti-Dlkantibodies, and conjugates of such fragments, and so-called "antigenbinding proteins" (single-chain antibodies) which are based on anti-Dlkantibodies, in accordance, for example, with U.S. Pat. No. 4,704,692,the contents of which are hereby incorporated by reference.Alternatively, Mabs or a fragment thereof within the present inventioncan be produced using conventional procedures via the expression ofisolated DNA which codes for variable regions of such an Mab in hostcells like E. coli, see, e.g., Ward et al., Nature 341: 544-546 (1989),or transfected murine myeloma cells. See Verhoyen et al., BioAssays 8:74 (1988); Gillies et al., Biotechnol. 7: 799-804 (1989); Nakatani etal., Biotechnol. 7:805-10 (1989).

Assays in which the above antibodies are employed can includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassays,immunoelectrophoresis, and the like. Also useful diagnostically areimmunohistochemical techniques which employ monoclonal antibodies ofknown, specific reactivities.

In accordance with this aspect of the present invention, a sample isobtained from a person to detect: (1) a small cell lung carcinoma, byremoving a body fluid or tissue suspected of harboring a tumor, such asalveolar, bronchiolar, or respiratory epithelial cells obtained from abronchial wash, nasopharyngeal aspirates, throat swabs or the like; (2)a metastasized neuroendocrine tumor, by biopsy, taken from tissue otherthan the adrenal gland (including cortex and medulla).Immuno-histochemical studies can be performed on such cells using amonoclonal antibody specific for Dlk.

Diagnostic applications of these antibodies are exemplified, accordingto the present invention, by the use of a kit containing an anti-Dlkantibody, which undergoes a reaction with a biological sample to detectDlk protein expression. Such a reaction involves the binding of anti-Dlkantibody to Dlk antigen, under conditions permissive of binding. Theobservation of an antibody-antigen complex in a biological sampleindicates a positive result. A kit of this sort could be used to detectthe extent of expression of Dlk in a particular biological sample froman individual, animal, or cell line.

Such an immunodiagnostic kit can include anti-Dlk antibody and areceptacle for containing the antibody in a sterilized form. The kit canfurther include anti-isotype serum antibody which recognizes theanti-Dlk antibody (Fc portion) and which is conjugated to a label, suchas an enzyme or fluorescent moiety.

In a preferred embodiment, a radiolabeled anti-Dlk antibody is provided.Such an antibody, preferably a monclonal antibody, is administered to ananimal or person for imaging purposes. After a suitable period of timefor the administered antibodies to bind Dlk expressing cells, a gammacamera machine is applied to detect the presence of labeled antibodieswithin the organism. Such a procedure provides information as to wherein the organism a primary or secondary Dlk-expressing neuroendocrinetumor is located.

A therapeutic application of anti-Dlk monoclonal antibodies includesadministration of anti-Dlk immunotoxins. Conjugation of an anti-Dlkmonoclonal antibody to a toxin, such as Psuedomonas exotoxin or othertoxins commonly conjugated to an antibody by means of a conventionalantibody-toxin linkage. Hertler et al., J. Clin. Oncol. 7(12): 1932(1989), describe methodologies for creating an antibody-toxin linkage,and is incorporated by reference herein. Thus, the anti-Dlk monoclonalantibody-toxin conjugates described are administered to an individual totarget and selectively kill dlk-expressing cells present inneuroendocrine tumors.

Similarly, a kit is provided which contains anti-Dlk immunotoxins in areceptacle. A kit can include the anti-Dlk immunotoxins and apharmaceutical excipient in a receptacle.

The present invention is further described with reference to thefollowing, illustrative examples. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art of theinvention. Although any methods and materials similar or equivalent tothose described herein can be used in the practice of the invention, thepreferred methods and materials have been described. Unless mentionedotherwise, the techniques employed or contemplated herein are standardmethodologies known to the art. The materials, methods and examples areillustrative only and not limiting.

Example 1 IDENTIFICATION OF dlk POLYNUCLEOTIDE AND POLYPEPTIDE MOLECULESIdentification of dlk

In investigating molecules associated with the gastrin-releasing peptide(GRP) responsive phenotype, s were identified which both were (1)expressed differentially between responsive murine Swiss andunresponsive murine Balb/c 3T3 fibroblasts, and (2) expressed inGRP-responsive human SCLC cell lines. The rationale for this approachwas that gene products correlated with a GRP-responsive phenotype wouldbe missing from Balb/c and unresponsive SCLC cell lines, but present inSwiss 3T3 fibroblasts and responsive SCLC cell lines.

A differential library was constructed that enriched for clonesexpressed in Swiss 3T3 but not in Balb/c 3T3 fibroblasts. Thedifferential library of Swiss 3T3 compared with Balb/c 3T3 fibroblastswas constructed as explained in detail in Timblin et al., Nucleic AcidsRes. 18: 1587 (1990). RNA isolation, electrophoresis, northern blots,and hybridization techniques were performed as described in Davis, etal., BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier, N.Y., (1986). Theprobes were labeled with ³² p dCTP (Amersham, Arlington Heights, Ill.)by the method of random primer as described in Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley and Sons, N.Y. 3.5.9-3.5.10(1991).

A partial length clone (150 nucleotides long) isolated from thisdifferential library hybridized with a 1.6 kilobase mRNA which showed anexpression pattern which matched with the two screening requirements.This partial length clone was used to screen a commercial oligodT-primed library of Swiss 3T3 fibroblasts in λZAPII vector (Stratagene(La Jolla, Calif.)), to obtain a full length clone.

Screening procedures and plasmid rescue of positive λZAPII clones wereperformed following the manufacturer's Stratagene) protocol, asdescribed by Short et al., Nuc. Acids Res. 16:7583 (1988). Severalclones with inserts around 1.6 kilobasepairs were obtained.

DNA Sequencing

Rescued plasmids were sequenced with Sequenase (USB, Cleveland, Ohio) bythe chain termination method, according to the manufacturer's protocoldescribed by Tabor et al., J. Biol. Chem. 214: 6447 (1989). Nucleotidesequence analysis of the s defined an open reading frame of 1155nucleotides, encoding a putative protein (Dlk) of 385 amino acids with amolecular weight of 41,320 daltons. This open reading frame wasclassified as coding by both Fickett's and Shepherd's methods. Fickettet al., Nucleic Acids Res. 10: 5303 (1982); Shepherd et al., Meth.Enzymol. 188:180 (1990). The open reading frames were identified bysoftware programs implementing these methods (PC/Gene software package,Intelligenetics Inc. (Mountain View, Calif.); A. Bairoch, Ph.D thesis,University of Geneva, (1990)).

In vitro Translation of Dlk Polypeptide

In vitro translation assays from mouse dlk mRNA were performed using arabbit reticulocyte lysate system from NEN (Boston, Mass.), according tomanufacturer's protocol, as described by Lockhard et al., Biochem.Biophys. Res. Comm. 37:204 (1969).

dlkmRNA was selected by hybridization of poly A⁺ RNA from Swiss 3T3fibroblasts with denatured full-length dlk immobilized on nitrocellulosefilters. (dlk mRNA was selected by hybridization of 2 μg of poly A⁺Swiss 3T3 RNA with 5 μg of nitrocellulose-immobilized denatured dlk TheRNA bound was eluted by boiling. Mouse dlk mRNA was also prepared invitro using two different full length dlk s cloned in pGEM4Z (Promega).These three mRNAs were used as templates for in vitro translation.

Labeled proteins were analyzed in a 12% polyacrylamide gel followed byfluorography. A protein band of around 42 kilodaltons was present in allthree samples, in agreement with the molecular weight of Dlkpolypeptide, predicted from its sequence.

A Comparison between mice and humans

Mouse and human dlk polynucleotide sequences are 86.2% identical and90.1% similarity in their amino acid sequence. They share many potentialsites of biological activity, including 6 EGF-like repeats (highlyhomologous to those found in invertebrate neurogenic proteins) anintegral transmembrane domain and a signal peptide domain.

The structural characteristics of dlk were analyzed with the programPC/Gene (Intelligenetics Inc. (Mountain View, Calif.), A. Bairoch, Ph.Dthesis, University of Geneva (1990)). The transmembrane domain was foundwith the program RAOARGOS, implementing the method of Rao and Argos,Biochim. Biophys. Acta 869: 197 (1986). The signal peptide was analyzedwith the program PSIGNAL, according to the method of Von Heijne, NucleicAcids Res. 14: 4683 (1986).

Example 2 A COMPARISON BETWEEN pPG2 and dlk GENE EXPRESSION IN MICE, &dlk GENE EXPRESSION IN HUMANS

In normal tissues of human, mouse and hamster origin, dlk expression wasdetected according to the present invention, only in adrenal andplacental tissue. Similarly, pG2 expression was known to be restrictedto adrenal gland in normal human tissues.

dlk mRNA was detected by Northern analysis in human and ratpheochromocytoma (PC12) cell lines. pG2 was identified inpheochromocytoma cell lines by Helman et al., PNAS USA 84:2336 (1987).

According to the present invention, dlk was detected in neuroblastoma(SK-N-SH) cells. pG2 expression in neuroblastoma cell lines was detectedin differentiated cells, but absent from undifferentiated neuroblastomacell lines. Cooper et al., Cell Growth and Diff. 1:149 (1989).

In addition, other cells which express dlk identified by the presentinvention include certain SCLC cell lines. Also, murine Swiss 3T3fibroblasts were found to express dlk, by using human dlk to probe underhigh stringency conditions. Balb 3T3 fibroblasts RNA were negative fordlk expression under these conditions.

To explore the relationship between mouse dlk and human pG2, s wereisolated and characterized from a λgt10 human adrenal gland library(Clontech, Palo Alto, Calif.) and screened according to themanufacturer's protocol, using mouse dlk as a hybridization probe. Evenunder low stringency conditions, no s were isolated which coded forproteins with structural characteristics similar to those reported forpG2. Positive λ clones were subcloned into PGEM4Z (Promega, MadisonWis.) and sequenced according to the method of Example 1. Sequence datafrom several full-length clones which were isolated indicated that theses showed a 82.1% sequence identity with mouse dlk and coded for thehuman counterpart of the mouse dlk protein (FIG., SEQ ID NOS:1 and 2).

Dlk's structural characterization is very different than that predictedfor pG2 protein (Helman et al., supra. (1987)), which consists of a 286amino acid sequence (about 30 kDa), contains no EGF-like repeats and nosignal peptide or transmembrane domains. This was so, despite a findingof an 81.2% nucleotide sequence identity of dlk with pG2 (adrenal glandcDNA library, Helman et al., supra (1990)), as determined usingnucleotide sequence homology analysis (GENBANK and EMBL databases). Itis assumed that pG2 is identified correctly as the dlk polynucleotidemolecule shown in FIG. 1.

Example 3 dlk/Dlk HOMOLOGY WITH OTHER GENES & PROTEINS

dlk shows a high degree of homology with the EGF-like neurogenic genesof Drosophila, which are involved in the decisions taken by the cells ofthe embryohal ectoderm to differentiate into epidermal or neuronalcells. Genes which were found to have highest homologyto Dlk include:Delta, Notch and Serrate of D. melanogaster, lin-12 and glp1 of C.elegans, and uEGF1 of the sea urchin. Although the degree of homologyvaried between the individual proteins and Dlk, regions of maximumhomology exhibited up to 33% amino acid identity, which rose to around75%, with allowance for conservative amino acid substitutions.

FIG. 4 shows the alignment of mouse or human dlk EGF-like repetitivesequences (SEQ ID NOS:6 and 7) with consensus sequences of EGF sequencerepeats (including SEQ ID NO:5) of several proteins. The alignment ofthe EGF-like repeats was done using the program CLUSTAL, described byHiggins et al., Gene 73: 237 (1988). The sites of potential biologicalimportance were analyzed with the program PROSITE. Residues wellconserved among homeotic genes also are conserved in dlk, confirming dlkas a member of the family of EGF-like homeotic genes. The amino acidsequence and structure of the EGF-like repeats, as well as the overallstructure of dlk, are more related to the invertebrate homeotic genesthan to other vertebrate non-homeotic EGF-like proteins, such asEGF-precursor, TGFα, the α, β1 and β2 chains of laminin, coagulationfactors, or complement proteins, previously thought to be the mammaliancounterparts of the invertebrate homeotic genes.

dlk gene was found present in species ranging from birds to human,including: yeast, Drosophila, Xenopus, mouse, rat, rabbit, chicken, dog,cow, monkey and human. However, despite the structural homology withinvertebrate proteins, dlk gene is absent from invertebrates and lowvertebrates.

The program PCOMPARE, described by Needleman et al., Mol. Biol. 48:443(1970), included in PC/Gene was used for analysis of homology. In thismethod, the optimal alignment score between two proteins were comparedwith the statistical distribution of 100 random alignments. An alignmentscore of greater than 5 positive standard deviations from the meanrandom alignment distribution was considered significant, particularlywhen no functional or structural relationship between the proteinscompared is known. Representative alignment scores were determined:Delta, 20.2; Serrate, 19.7; TAN-1, 16.2; Notch, 14.6; Xotch, 13.6;Drosophila Laminin β2, 6.3; mouse Laminin β2, 4.1; human coagulationfactor XII, 2.8; and human EGF precursor, 0.6.

Example 4 dlk EXPRESSION: NORTHERN BLOT mRNA ANALYSIS

Expression of dlk was detected by Northern analysis in SCLC linesNCI-H510, NCI-H69 and NCI-N592; in human neuroblastoma line SK-N-SH, andin the rat pheochromocytoma PC-12 cell line. Twenty μg of total RNA or 2μg of poly A⁺ were run in a 1% agarose gel and then blotted on anitrocellulose filter (described in Ex. 1).

A 1.6 Kb band corresponding to dlk was observed only in the SCLC celllines NCI-N592, NCI-H69 and NCI-H510, and in Swiss 3T3 fibroblasts.Mouse Swiss 3T3 fibroblast RNA also showed a high degree of expressionof dlk, even if the hybridization was performed at high stringency withhuman dlk as a probe. Similar results were obtained using mouse dlk as aprobe. Balb/c 3T3 fibroblast RNA was negative for dlk expression underthese conditions. Ewing's sarcoma cell lines SK-ES-1, A4573 and TC106did not express dlk.

In normal tissues of mouse, hamster, and human origin dlk expression wasdetected exclusively in the adrenal gland.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 385 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: Mouse Dlk                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetIleAlaThrGlyAlaLeuLeuArgValLeuLeuLeuLeuLeuAla                              151015                                                                        PheGlyHisSerThrTyrGlyAlaGluCysAspProProCysAspPro                              202530                                                                        GlnTyrGlyPheCysGluAlaAspAsnValCysArgCysHisValGly                              354045                                                                        TrpGluGlyProLeuCysAspLysCysValThrAlaProGlyCysVal                              505560                                                                        AsnGlyValCysLysGluProTrpGlnCysIleCysLysAspGlyTrp                              65707580                                                                      AspGlyLysPheCysGluIleAspValArgAlaCysThrSerThrPro                              859095                                                                        CysAlaAsnAsnGlyThrCysValAspLeuGluLysGlyGlnTyrGlu                              100105110                                                                     CysSerCysThrProGlyPheSerGlyLysAspCysGlnHisLysAla                              115120125                                                                     GlyProCysValIleAsnGlySerProCysGlnHisGlyGlyAlaCys                              130135140                                                                     ValAspAspGluGlyGlnAlaSerHisAlaSerCysLeuCysProPro                              145150155160                                                                  GlyPheSerGlyAsnPheCysGluIleValAlaAlaThrAsnSerCys                              165170175                                                                     ThrProAsnProCysGluAsnAspGlyValCysThrAspIleGlyGly                              180185190                                                                     AspPheArgCysArgCysProAlaGlyPheValAspLysThrCysSer                              195200205                                                                     ArgProValSerAsnCysAlaSerGlyProCysGlnAsnGlyGlyThr                              210215220                                                                     CysLeuGlnHisThrGlnValSerPheGluCysLeuCysLysProPro                              225230235240                                                                  PheMetGlyProThrCysAlaLysLysArgGlyAlaSerProValGln                              245250255                                                                     ValThrHisLeuProSerGlyTyrGlyLeuThrTyrArgLeuThrPro                              260265270                                                                     GlyValHisGluLeuProValGlnGlnProGluGlnHisIleLeuLys                              275280285                                                                     ValSerMetLysGluLeuAsnLysSerThrProLeuLeuThrGluGly                              290295300                                                                     GlnAlaIleCysPheThrIleLeuGlyValLeuThrSerLeuValVal                              305310315320                                                                  LeuGlyThrValAlaIleValPheLeuAsnLysCysGluThrTrpVal                              325330335                                                                     SerAsnLeuArgTyrAsnHisThrPheArgLysLysLysAsnLeuLeu                              340345350                                                                     LeuGlnTyrAsnSerGlyGluGluLeuAlaValAsnIleIlePhePro                              355360365                                                                     GluLysIleAspMetThrThrPheAsnLysGluAlaGlyAspGluGlu                              370375380                                                                     Ile                                                                           385                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 383 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: Human Dlk                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetThrAlaThrGluAlaLeuLeuArgValLeuLeuLeuLeuLeuAla                              151015                                                                        PheGlyHisSerThrTyrGlyAlaGluCysPheProAlaCysAsnPro                              202530                                                                        GlnAsnGlyPheCysGluAspAspAsnValCysArgCysGlnProGly                              354045                                                                        TrpGlnGlyProLeuCysAspGlnCysValThrSerProGlyCysLeu                              505560                                                                        HisGlyLeuCysGlyGluProGlyGlnCysIleCysThrAspGlyTrp                              65707580                                                                      AspGlyGluLeuCysAspArgAspValArgAlaCysSerSerAlaPro                              859095                                                                        CysAlaAsnAsnGlyThrCysValSerLeuAspAspGlyLeuTyrGlu                              100105110                                                                     CysSerCysAlaProGlyTyrSerGlyLysAspCysGlnLysLysAsp                              115120125                                                                     GlyProCysValIleAsnGlySerProCysGlnHisGlyGlyThrCys                              130135140                                                                     ValAspAspGluGlyArgAlaSerHisAlaSerCysLeuCysProPro                              145150155160                                                                  GlyPheSerGlyAsnPheCysGluIleValAlaAsnSerCysThrPro                              165170175                                                                     AsnProCysGluAsnAspGlyValCysThrAspIleGlyGlyAspPhe                              180185190                                                                     ArgCysArgCysProAlaGlyPheIleAspLysThrCysSerArgPro                              195200205                                                                     ValThrAsnCysAlaSerSerProCysGlnAsnGlyGlyThrCysLeu                              210215220                                                                     GlnHisThrGlnValSerTyrGluCysLeuCysLysProGluPheThr                              225230235240                                                                  GlyLeuThrCysValLysLysArgAlaLeuSerProGlnGlnValThr                              245250255                                                                     ArgLeuProSerGlyTyrGlyLeuAlaTyrArgLeuThrProGlyVal                              260265270                                                                     HisGluLeuProValGlnGlnProGluHisArgIleLeuLysValSer                              275280285                                                                     MetLysGluLeuAsnLysLysThrProLeuLeuThrGluGlyGlnAla                              290295300                                                                     IleCysPheThrIleLeuGlyValLeuThrSerLeuValValLeuGly                              305310315320                                                                  ThrValGlyIleValPheLeuAsnLysCysGluThrTrpValSerAsn                              325330335                                                                     LeuArgTyrAsnHisMetLeuArgLysLysLysAsnLeuLeuLeuGln                              340345350                                                                     TyrAsnSerGlyGluAspLeuAlaValAsnIleIlePheProGluLys                              355360365                                                                     IleAspMetThrThrPheSerLysGluAlaGlyAspGluGluIle                                 370375380                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1556 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: Human Dlk                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TCTAAAGGAGGTGGAGAGCGCACCGCAGCCCGGTGCAGCCCGGTGCAGCCCTGGCTTTCC60                CCTCGCTGCGGCCCGTGCCCCCTTTCGCGTCCGCAACCAGAAGCCCAGTGCGGCGCCAGG120               AGCCGGACCCGCGCCCGCACCGCTCCCGGGACCGCGACCCCGGCCGCCCAGAGATGACCG180               CGACCGAAGCCCTCCTGCGCGTCCTCTTGCTCCTGCTGGCTTTCGGCCACAGCACCTATG240               GGGCTGAATGCTTCCCGGCCTGCAACCCCCAAAATGGATTCTGCGAGGATGACAATGTTT300               GCAGGTGCCATGTCGGCTGGCAGGGTCCCCTTTGTGACCAGTGCGTGACCTCTCCCGGCT360               GCCTTCACGGACTCTGTGGAGAACCCGGGCAGTGCATTTGCACCGACGGCTGGGACGGGG420               AGCTCTGTGATAGAGATGTTCGGGCCTGCTCCTCGGCCCCCTGTGCCAACAACGGGACCT480               GCGTGAGCCTGGACGGTGGCCTCTATGAATGCTCCTGTGCCCCCGGGTACTCGGGAAAGG540               ACTGCCAGAAAAAGGACGGGCCCTGTGTGATCAACGGCTCCCCCTGCCAGCACGGAGGCA600               CCTGCGTGGATGATGAGGGCCGGGCCTCCCATGCCTCCTGCCTGTGCCCCCCTGGCTTCT660               CAGGCAATTTCTGCGAGATCGTGGCCAACAGCTGCACCCCCAACCCATGCGAGAACGACG720               GCGTCTGCACTGACATTGGGGGCGACTTCCGCTGCCGGTGCCCAGCCGGCTTCATCGACA780               AGACCTGCAGCCGCCCGGTGACCAACTGCGCCAGCAGCCCGTGCCAGAACGGGGGCACCT840               GCCTGCAGCACACCCAGGTGAGCTACGAGTGTCTGTGCAAGCCCGAGTTCACAGGTCTCA900               CCTGTGTCAAGAAGCGCGCGCTGAGCCCCCAGCAGGTCACCCGTCTGCCCAGCGGCTATG960               GGCTGGCCTACCGCCTGACCCCTGGGGTGCACGAGCTGCCGGTGCAGCAGCCGGAGCACC1020              GCATCCTGAAGGTGTCCATGAAAGAGCTCAACAAGAAAACCCCTCTCCTCACCGAGGGCC1080              AGGCCATCTGCTTCACCATCCTGGGCGTGCTCACCAGCCTGGTGGTGCTGGGCACTGTGG1140              GTATCGTCTTCCTCAACAAGTGCGAGACCTGGGTGTCCAACCTGCGCTACAACCACATGC1200              TGCGGAAGAAGAAGAACCTGCTGCTTCAGTACAACAGCGGGGAGGACCTGGCCGTCAACA1260              TCATCTTCCCCGAGAAGATCGACATGACCACCTTCAGCAAGGAGGCCGGCGACGAGGAGA1320              TCTAAGCAGCGTTCCCACAGCCCCCTCTAGATTCTTGGAGTTCCGCAGAGCTTACTATAC1380              GCGGTCTGTCCTAATCTTTGTGGTGTTCGCTATCTCTTGTGTCAAATCTGGTGAACGCTA1440              CGCTTACATATATTGTCTTTGTGCTGCTGTGTGACAAACGCAATGCAAAAACAATCCTCT1500              TTCTCTCTCTTAATGCATGATACAGAATAATAATAAGAATTTCATCTTTAAATGAG1556                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1573 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (vi) ORIGINAL SOURCE:                                                         (B) STRAIN: Mouse Dlk                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GGTGCAACCCTAGCTTTCTTCCCGCTGGACGCCCGTGCCCCCTTCGTGGTCCGCAACCAG60                AAGCCCAGCGCACGCCCCGGAGCAGCCCCTGCACCGCCTCCGCTCCCCGGACCGCGACCC120               AGGCCGCCCCGAGATGATCGCGACCGGAGCCCTCCTGCGCGTCCTCTTGCTCCTGCTGGC180               TTTCGGCCACAGCACCTATGGGGCTGAATGCGACCCACCCTGTGACCCCCAGTATGGATT240               CTGCGAGGCTGACAATGTCTGCAGGTGCCATGTTGGCTGGGAGGGTCCCCTCTGTGACAA300               GTGTGTAACTGCCCCTGGCTGTGTCAATGGAGTCTGCAAGGAACCATGGCAGTGCATCTG360               CAAGGATGGCTGGGACGGGAAATTCTGCGAAATAGACGTTCGGGCTTGCACCTCAACCCC420               CTGCGCCAACAATGGAACTTGCGTGGACCTGGAGAAAGGCCAGTACGAATGCTCCTGCAC480               ACCTGGGTTCTCTGGAAAGGACTGCCAGCACAAGGCTGGGCCCTGCGTGATCAATGGTTC540               TCCCTGCCAGCACGGAGGCGCCTGCGTGGATGATGAGGGCCAGGCCTCGCATGCTTCCTG600               CCTGTGCCCCCCTGGCTTCTCAGGCAACTTCTGTGAGATCGTAGCCGCAACCAACAGCTG660               TACCCCTAACCCATGCGAGAACGATGGCGTCTGCACCGACATCGGGGGTGACTTCCGTTG720               CCGCTGCCCAGCTGGATTCGTCGACAAGACCTGCAGCCGCCCGGTGAGCAACTGCGCCAG780               TGGCCCGTGCCAGAACGGGGGCACCTGCCTCCAGCACACCCAGGTGAGCTTCGAGTGTCT840               GTGCAAGCCCCCGTTCATGGGTCCCACGTGCGCGAAGAAGCGCGGGGCTAGCCCCGTGCA900               GGTCACCCACCTGCCCAGCGGCTATGGGCTCACCTACCGCCTGACCCCCGGGGTGCACGA960               GCTGCCTGTTCAGCAGCCCGAGCAACACATCCTGAAGGTGTCCATGAAAGAGCTCAACAA1020              GAGTACCCCTCTCCTCACCGAGGGACAGGCCATCTGCTTCACCATCCTGGGCGTGCTCAC1080              CAGCCTGGTGGTGCTGGGCACCGTGGCCATCGTCTTTCTCAACAAGTGCGAAACCTGGGT1140              GTCCAACCTGCGCTACAACCACACGTTTCGCAAGAAGAAGAACCTCCTGTTGCAGTATAA1200              CAGCGGCGAGGAGCTGGCGGTCAATATCATCTTCCCCGAGAAGATTGACATGACCACCTT1260              CAACAAGGAGGCTGGTGATGAGGAGATCTAAGCAGCGTTCCCCACCCCCACTCCCAGGCC1320              CTTCACCCCGACCCCGACCCAGGCCCTCTCTATTACCGGGTTCCTTTAGAGCTCTCTACC1380              GAGTCTGGCTTTTTGTGGTGGAGTTTGCTCTATTGTGTGGAATCGAGTGAAGCCTATGCT1440              TACATATATTGTCTTGTGTTGCTGTGTGCCATGCTACCTCGCTATCTAAGAACCCCTTCC1500              TCCCTATTAATGCATGATAATGAATAATAATAATAAGAATTTCATCTCTAAATGAAAAAA1560              AAAAAAAAAAAAG1573                                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AsnGlyGlyThrCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysProSerSerTyrAspGlyTyrCysLeuAsnGlyGlyValCysMet                              151015                                                                        HisIleGluSerAspLeuSerTyrThr                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CysAsnCysValIleGlyTyrSerGlyAspArgCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CAAGCCCGAGTTCACAGGTC20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCGGGGAAGATGTTGAC17                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated Dlk polypeptide consistingessentially of the amino acid sequence shown in FIG. 1B (SEQ ID NO:2).